Dark fermentation of pretreated hydrolysates of pineapple fruit waste for the production of biohydrogen using bacteria isolated from wastewater sources.

In the present study, both acidic and alkaline hydrolysate of pineapple waste was utilised for the production of biohydrogen using locally isolated bacterial strains. The bacteria were isolated from different wastewater sources and were identified as Proteus mirabilis, Pseudomonas aeruginosa, Bacillus altitudinus, Bacillus subtilis, Paenibacillus alvei, and Lysinibacillus sphaericus. Experimental results showed that the highest biohydrogen yield of 836.33 ± 48.02 mL H2 was produced from alkaline hydrolysate with Bacillus altitudinis during the 96thhr of fermentation. Among the different bacterial strains, B. altitudinis showed higher H2 production. Comparatively alkaline hydrolysates exhibited a higher yield of hydrogen than acidic hydrolysates. The final pH of the experiment was found to be in acidic range. The total VFA concentration ranged between 930 ± 207.85 mg/L to 3050 ± 476.97 mg/L. Both sugar degradation and COD reduction were more than 80% in the acidic and alkaline hydrolysates while the lowest sugar degradation and COD reduction were observed for the untreated biomass. The rationale behind this study was to convert the waste biomass into energy by utilising the potential of native bacterial communities.

Genome-Wide Identification and Characterization of Gibberellic Acid-Stimulated Arabidopsis Gene Family in Pineapple (Ananas comosus).

The gibberellic acid-stimulated Arabidopsis (GASA) gene family plays a crucial role in growth, development, and stress response, and it is specific to plants. This gene family has been extensively studied in various plant species, and its functional role in pineapple has yet to be characterized. In this study, 15 AcGASA genes were identified in pineapple through a genome-wide scan and categorized into three major branches based on a phylogenetic tree. All AcGASA proteins share a common structural domain with 12 cysteine residues, but they exhibit slight variations in their physicochemical properties and motif composition. Predictions regarding subcellular localization suggest that AcGASA proteins are present in the cell membrane, Golgi apparatus, nucleus, and cell wall. An analysis of gene synteny indicated that both tandem and segmental repeats have a significant impact on the expansion of the AcGASA gene family. Our findings demonstrate the differing regulatory effects of these hormones (GA, NAA, IAA, MeJA, and ABA) on the AcGASA genes. We analyzed the expression profiles of GASA genes in different pineapple tissue parts, and the results indicated that AcGASA genes exhibit diverse expression patterns during the development of different plant tissues, particularly in the regulation of floral organ development. This study provides a comprehensive understanding of GASA family genes in pineapple. It serves as a valuable reference for future studies on the functional characterization of GASA genes in other perennial herbaceous plants.

Integrative Analysis of Metabolome and Transcriptome Provides Insights into the Mechanism of Flower Induction in Pineapple (Ananas comosus (L.) Merr.) by Ethephon.

Exogenous ethylene is commonly utilized to initiate flower induction in pineapple (Ananas comosus (L.) Merr.). However, the molecular mechanisms and metabolic changes involved are not well understood. In this study, we explored the genetic network and metabolic shifts in the 'Comte de Paris' pineapple variety during ethylene-induced flowering. This was achieved through an integrative analysis of metabolome and transcriptome profiles at vegetative shoot apexes (0 d after ethephon treatment named BL_0d), the stage of bract primordia (8 d after ethephon treatment named BL_8d), stage of flower primordia (18 d after ethephon treatment named BL_18d), and the stage of stopped floret differentiation (34 d after ethephon treatment named BL_34d). We isolated and identified 804 metabolites in the pineapple shoot apex and inflorescence, categorized into 24 classes. Notably, 29, 31, and 46 metabolites showed significant changes from BL_0d to BL_8d, BL_8d to BL_18d, and BL_18d to BL_34d, respectively. A marked decrease in indole was observed, suggesting its role as a characteristic metabolite during flower induction. Transcriptomic analysis revealed 956, 1768, and 4483 differentially expressed genes (DEGs) for BL_0d vs. BL_8d, BL_8d vs. BL_18d, and BL_18d vs. BL_34d, respectively. These DEGs were significantly enriched in carbohydrate metabolism and hormone signaling pathways, indicating their potential involvement in flower induction. Integrating metabolomic and transcriptomic data, we identified several candidate genes, such as Agamous-Like9 (AGL9), Ethylene Insensitive 3-like (ETIL3), Apetala2 (AP2), AP2-like ethylene-responsive transcription factor ANT (ANT), and Sucrose synthase 2 (SS2), that play potentially crucial roles in ethylene-induced flower induction in pineapple. We also established a regulatory network for pineapple flower induction, correlating metabolites and DEGs, based on the Arabidopsis thaliana pathway as a reference. Overall, our findings offer a deeper understanding of the metabolomic and molecular mechanisms driving pineapple flowering.

Integration of Metabolomics and Transcriptomics to Explore Dynamic Alterations in Fruit Color and Quality in 'Comte de Paris' Pineapples during Ripening Processes.

Pineapple color yellowing and quality promotion gradually manifest as pineapple fruit ripening progresses. To understand the molecular mechanism underlying yellowing in pineapples during ripening, coupled with alterations in fruit quality, comprehensive metabolome and transcriptome investigations were carried out. These investigations were conducted using pulp samples collected at three distinct stages of maturity: young fruit (YF), mature fruit (MF), and fully mature fruit (FMF). This study revealed a noteworthy increase in the levels of total phenols and flavones, coupled with a concurrent decline in lignin and total acid contents as the fruit transitioned from YF to FMF. Furthermore, the analysis yielded 167 differentially accumulated metabolites (DAMs) and 2194 differentially expressed genes (DEGs). Integration analysis based on DAMs and DEGs revealed that the biosynthesis of plant secondary metabolites, particularly the flavonol, flavonoid, and phenypropanoid pathways, plays a pivotal role in fruit yellowing. Additionally, RNA-seq analysis showed that structural genes, such as FLS, FNS, F3H, DFR, ANR, and GST, in the flavonoid biosynthetic pathway were upregulated, whereas the COMT, CCR, and CAD genes involved in lignin metabolism were downregulated as fruit ripening progressed. APX as well as PPO, and ACO genes related to the organic acid accumulations were upregulated and downregulated, respectively. Importantly, a comprehensive regulatory network encompassing genes that contribute to the metabolism of flavones, flavonols, lignin, and organic acids was proposed. This network sheds light on the intricate processes that underlie fruit yellowing and quality alterations. These findings enhance our understanding of the regulatory pathways governing pineapple ripening and offer valuable scientific insight into the molecular breeding of pineapples.

Identification of bromelain subfamily proteases encoded in the pineapple genome.

Papain (aka C1A) family proteases, including bromelain enzymes, are widespread across the plant kingdom and play critical regulatory functions in protein turnover during development. The proteolytic activity exhibited by papain family proteases has led to their increased usage for a wide range of cosmetic, therapeutic, and medicinal purposes. Bromelain enzymes, or bromelains in short, are members of the papain family that are specific to the bromeliad plant family. The only major commercial extraction source of bromelain is pineapple. The importance of C1A family and bromelain subfamily proteases in pineapple development and their increasing economic importance led several researchers to utilize available genomic resources to identify protease-encoding genes in the pineapple genome. To date, studies are lacking in screening bromelain genes for targeted use in applied science studies. In addition, the bromelain genes coding for the enzymes present in commercially available bromelain products have not been identified and their evolutionary origin has remained unclear. Here, using the newly developed MD2 v2 pineapple genome, we aimed to identify bromelain-encoding genes and elucidate their evolutionary origin. Orthologous and phylogenetic analyses of all papain-family proteases encoded in the pineapple genome revealed a single orthogroup (189) and phylogenetic clade (XIII) containing the bromelain subfamily. Duplication mode and synteny analyses provided insight into the origin and expansion of the bromelain subfamily in pineapple. Proteomic analysis identified four bromelain enzymes present in two commercially available bromelain products derived from pineapple stem, corresponding to products of four putative bromelain genes. Gene expression analysis using publicly available transcriptome data showed that 31 papain-family genes identified in this study were up-regulated in specific tissues, including stem, fruit, and floral tissues. Some of these genes had higher expression in earlier developmental stages of different tissues. Similar expression patterns were identified by RT-qPCR analysis with leaf, stem, and fruit. Our results provide a strong foundation for future applicable studies on bromelain, such as transgenic approaches to increase bromelain content in pineapple, development of bromelain-producing bioreactors, and studies that aim to determine the medicinal and/or therapeutic viability of individual bromelain enzymes.

The Synergistic Mechanism of Photosynthesis and Antioxidant Metabolism between the Green and White Tissues of Ananas comosus var. bracteatus Chimeric Leaves.

Ananas comosus var. bracteatus (Ac. bracteatus) is a typical leaf-chimeric ornamental plant. The chimeric leaves are composed of central green photosynthetic tissue (GT) and marginal albino tissue (AT). The mosaic existence of GT and AT makes the chimeric leaves an ideal material for the study of the synergistic mechanism of photosynthesis and antioxidant metabolism. The daily changes in net photosynthetic rate (NPR) and stomatal conductance (SCT) of the leaves indicated the typical crassulacean acid metabolism (CAM) characteristic of Ac. bracteatus. Both the GT and AT of chimeric leaves fixed CO2 during the night and released CO2 from malic acid for photosynthesis during the daytime. The malic acid content and NADPH-ME activity of the AT during the night was significantly higher than that of GT, which suggests that the AT may work as a CO2 pool to store CO2 during the night and supply CO2 for photosynthesis in the GT during the daytime. Furthermore, the soluble sugar content (SSC) in the AT was significantly lower than that of GT, while the starch content (SC) of the AT was apparently higher than that of GT, indicating that AT was inefficient in photosynthesis but may function as a photosynthate sink to help the GT maintain high photosynthesis activity. Additionally, the AT maintained peroxide balance by enhancing the non-enzymatic antioxidant system and antioxidant enzyme system to avoid antioxidant damage. The enzyme activities of reductive ascorbic acid (AsA) and the glutathione (GSH) cycle (except DHAR) and superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were enhanced, apparently to make the AT grow normally. This study indicates that, although the AT of the chimeric leaves was inefficient at photosynthesis because of the lack of chlorophyll, it can cooperate with the GT by working as a CO2 supplier and photosynthate store to enhance the photosynthetic ability of GT to help chimeric plants grow well. Additionally, the AT can avoid peroxide damage caused by the lack of chlorophyll by enhancing the activity of the antioxidant system. The AT plays an active role in the normal growth of the chimeric leaves.

Integrated Metabolome and Transcriptome Analysis Reveals a Potential Mechanism for Water Accumulation Mediated Translucency in Pineapple (Ananas comosus (L.) Merr.) Fruit.

A physiological disease of the pineapple fruit called pineapple translucency causes the pulp to become water-soaked, which affects the fruit's taste, flavor, shelf life, and integrity. In the present study, we analyzed seven pineapple varieties, of which three were watery and four were non-watery. There were no apparent macronutritional (K, P, or N) differences in their pulp, but the non-watery pineapple varieties had higher dry matter and soluble sugar content. The metabolomic analysis found 641 metabolites and revealed differential expression of alkaloids, phenolic acids, nucleotide derivatives, lipids, and other metabolites among the seven species. Transcriptome analysis and further KEGG enrichment showed downregulation of 'flavonoid biosynthesis' pathways, differential expression of metabolic pathways, secondary metabolites biosynthesis, plant-pathogen interaction, and plant hormone signal transduction. We believe this study will provide critical molecular data supporting a deeper understanding of pineapple translucency formation and greatly benefit future research on this commercially important crop.

Function of ALA Content in Porphyrin Metabolism Regulation of Ananas comosus var. bracteatus.

Chlorophyll and heme are essential molecules for photosynthesis and respiration, which are competing branches of the porphyrin metabolism pathway. Chlorophyll and heme balance regulation is very important for the growth and development of plants. The chimeric leaves of Ananas comosus var. bracteatus were composed of central photosynthetic tissue (PT) and marginal albino tissue (AT), which were ideal materials for the study of porphyrin metabolism mechanisms. In this study, the regulatory function of ALA content on porphyrin metabolism (chlorophyll and heme balance) was revealed by comparing PT and AT, 5-Aminolevulinic Acid (ALA) exogenous supply, and interference of hemA expression. The AT remained similar in porphyrin metabolism flow level to the PT by keeping an equal ALA content in both tissues, which was very important for the normal growth of the chimeric leaves. As the chlorophyll biosynthesis in AT was significantly inhibited, the porphyrin metabolism flow was directed more toward the heme branch. Both tissues had similar Mg2+ contents; however, Fe2+ content was significantly increased in the AT. The chlorophyll biosynthesis inhibition in the white tissue was not due to a lack of Mg2+ and ALA. A 1.5-fold increase in ALA content inhibited chlorophyll biosynthesis while promoting heme biosynthesis and hemA expression. The doubling of ALA content boosted chlorophyll biosynthesis while decreasing hemA expression and heme content. HemA expression interference resulted in a higher ALA content and a lower chlorophyll content, while the heme content remained at a relatively low and stable level. Conclusively, a certain amount of ALA was important for the stability of porphyrin metabolism and the normal growth of plants. The ALA content appears to be able to regulate chlorophyll and heme content by bidirectionally regulating porphyrin metabolism branch direction.

Small Auxin Up RNA (SAUR) gene family identification and functional genes exploration during the floral organ and fruit developmental stages in pineapple (Ananas comosus L.) and its response to salinity and drought stresses.

In plants, sexual reproduction relies on the proper development of floral organs that facilitate the successful development of fruits and seeds. Auxin responsive small auxin-up RNA (SAUR) genes play essential roles in floral organ formation and fruit development. However, little is known about the role of SAUR genes in pineapple floral organ formation and fruit development as well as stress responses. In this study, based on genome information and transcriptome datasets, 52 AcoSAUR genes were identified and grouped into 12 groups. The gene structure analysis revealed that most AcoSAUR genes did not have introns, although auxin-acting elements were abundant in the promoter region of AcoSAUR members. The expression analysis across the multiple flower and fruit development stages revealed differential expression of AcoSAUR genes, indicating a tissue and stage-specific function of AcoSAURs. Correlation analysis and pairwise comparisons between gene expression and tissue specificity identified stamen-, petal-, ovule-, and fruit-specific AcoSAURs involved in pineapple floral organs (AcoSAUR4/5/15/17/19) and fruit development (AcoSAUR6/11/36/50). RT-qPCR analysis revealed that AcoSAUR12/24/50 played positive roles in response to the salinity and drought treatment. This work provides an abundant genomic resource for functional analysis of AcoSAUR genes during the pineapple floral organs and fruit development stages. It also highlights the role of auxin signaling involved in pineapple reproductive organ growth.

Unveiling the molecular mechanism involving anthocyanins in pineapple peel discoloration during fruit maturation.

Peel color is a key factor that affects the fruit's aesthetic and economic values. Limited knowledge is available on the regulation of pineapple peel discoloration. Here, we report that a decrease in anthocyanin biosynthesis, particularly cyanidin, is predominantly associated with the pineapple peel color change during maturation. The findings suggest that the changes in the expression of key structural genes (early and late biosynthetic genes) of the anthocyanin (cyanidin) biosynthesis pathway are responsible for peel discoloration. Based on a gene co-expression analysis and a transient expression, two transcription factors i.e., AcHOX21 and AcMYB12, were identified, whose' downregulation leads to reduced anthocyanin accumulation with fruit maturation. The endogenous levels of jasmonic acid, gibberellic acid, and auxins are also involved in anthocyanin-content-led peel discoloration. Overall, the discovery of genes regulating anthocyanin biosynthesis in pineapple peel provides a theoretical basis for improving the fruit's aesthetic value through genetic engineering.

Genome-Wide Identification and Characterization of R2R3-MYB Provide Insight into Anthocyanin Biosynthesis Regulation Mechanism of Ananas comosus var. bracteatus.

The R2R3-MYB proteins comprise the largest class of MYB transcription factors, which play an essential role in regulating anthocyanin synthesis in various plant species. Ananas comosus var. bracteatus is an important colorful anthocyanins-rich garden plant. The spatio-temporal accumulation of anthocyanins in chimeric leaves, bracts, flowers, and peels makes it an important plant with a long ornamental period and highly improves its commercial value. We conducted a comprehensive bioinformatic analysis of the R2R3-MYB gene family based on genome data from A. comosus var. bracteatus. Phylogenetic analysis, gene structure and motif analysis, gene duplication, collinearity, and promoter analysis were used to analyze the characteristics of this gene family. In this work, a total of 99 R2R3-MYB genes were identified and classified into 33 subfamilies according to phylogenetic analysis, and most of them were localized in the nucleus. We found these genes were mapped to 25 chromosomes. Gene structure and protein motifs were conserved among AbR2R3-MYB genes, especially within the same subfamily. Collinearity analysis revealed four pairs of tandem duplicated genes and 32 segmental duplicates in AbR2R3-MYB genes, indicating that segmental duplication contributed to the amplification of the AbR2R3-MYB gene family. A total of 273 ABRE responsiveness, 66 TCA elements, 97 CGTCA motifs, and TGACG motifs were the main cis elements in the promoter region under response to ABA, SA, and MEJA. These results revealed the potential function of AbR2R3-MYB genes in response to hormone stress. Ten R2R3-MYBs were found to have high homology to MYB proteins reported to be involved in anthocyanin biosynthesis from other plants. RT-qPCR results revealed the 10 AbR2R3-MYB genes showed tissue-specific expression patterns, six of them expressed the highest in the flower, two genes in the bract, and two genes in the leaf. These results suggested that these genes may be the candidates that regulate anthocyanin biosynthesis of A. comosus var. bracteatus in the flower, leaf, and bract, respectively. In addition, the expressions of these 10 AbR2R3-MYB genes were differentially induced by ABA, MEJA, and SA, implying that these genes may play crucial roles in hormone-induced anthocyanin biosynthesis. Our study provided a comprehensive and systematic analysis of AbR2R3-MYB genes and identified the AbR2R3-MYB genes regulating the spatial-temporal anthocyanin biosynthesis in A. comosus var. bracteatus, which would be valuable for further study on the anthocyanin regulation mechanism of A. comosus var. bracteatus.

Genome-Wide Identification and Abiotic Stress Response Analysis of PP2C Gene Family in Woodland and Pineapple Strawberries.

Protein phosphatase 2C (PP2C) is a negative regulator of serine/threonine residue protein phosphatase and plays an important role in abscisic acid (ABA) and abiotic-stress-mediated signaling pathways in plants. The genome complexity of woodland strawberry and pineapple strawberry is different due to the difference in chromosome ploidy. This study conducted a genome-wide investigation of the FvPP2C (Fragaria vesca) and FaPP2C (Fragaria ananassa) gene family. Fifty-six FvPP2C genes and 228 FaPP2C genes were identified from the woodland strawberry and pineapple strawberry genomes, respectively. FvPP2Cs were distributed on seven chromosomes, and FaPP2Cs were distributed on 28 chromosomes. The size of the FaPP2C gene family was significantly different from that of the FvPP2C gene family, but both FaPP2Cs and FvPP2Cs were localized in the nucleus, cytoplasm, and chloroplast. Phylogenetic analysis revealed that 56 FvPP2Cs and 228 FaPP2Cs could be divided into 11 subfamilies. Collinearity analysis showed that both FvPP2Cs and FaPP2Cs had fragment duplication, and the whole genome duplication was the main cause of PP2C gene abundance in pineapple strawberry. FvPP2Cs mainly underwent purification selection, and there were both purification selection and positive selection effects in the evolution of FaPP2Cs. Cis-acting element analysis found that the PP2C family genes of woodland and pineapple strawberries mainly contained light responsive elements, hormone responsive elements, defense and stress responsive elements, and growth and development-related elements. The results of quantitative real-time PCR (qRT-PCR) showed that the FvPP2C genes showed different expression patterns under ABA, salt, and drought treatment. The expression level of FvPP2C18 was upregulated after stress treatment, which may play a positive regulatory role in ABA signaling and abiotic stress response mechanisms. This study lays a foundation for further investigation on the function of the PP2C gene family.

Low temperature storage alleviates internal browning of 'Comte de Paris' winter pineapple fruit by reducing phospholipid degradation, phosphatidic acid accumulation and membrane lipid peroxidation processes.

To uncover the mechanism underlying membrane lipid metabolism of low temperature induced internal browning tolerance in pineapple, membrane phospholipid alterations of harvested 'Comte de Paris' winter pineapple fruit stored at either 10 °C or 25 °C was investigated. Fruit stored at 10 °C developed low levels of internal browning as compared to fruit stored at 25 °C and was associated with high contents of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine, and low levels of phosphatidic acid. Storage at 10 °C down-regulated the expression levels of phospholipase As. Fruit stored at 10 °C also exhibited high ratio of unsaturated fatty acid to saturated fatty acid and index of unsaturated fatty acid level. These findings suggest that maintenance phospholipid abundance, reduction in phosphatidic acid accumulation and membrane lipid peroxidation may have contributed to the enhanced internal browning tolerance in 'Comte de Paris' winter pineapple fruit at low temperature storage.

Implications of nasal delivery of bromelain on its pharmacokinetics, tissue distribution and pharmacodynamic profile-A preclinical study.

Asthma is a polygenic chronic inflammatory respiratory disease devastating the quality of life and state economies. Therefore, utilization of natural products as a therapeutic approach has attained wider consideration for development of novel drugs for asthma management. Bromelain, a mixture of natural bioactive cysteine proteases abundantly found in pineapple stem, has allured attention for its pharmacological activities. However, poor stability in gastric milieu, high dose and immunogenicity associated with prolonged use hinders its oral use. Therefore, need exists to explore alternative route of bromelain administration to achieve its plausible benefits. The present study investigated the preclinical prospects of nasal administration of bromelain on systemic bioavailability, tissue distribution and it's in vivo anti-histaminic, bronchodilator and anti-asthmatic activity in animal models. Pharmacokinetic studies revealed 1.43-fold higher relative bioavailability with faster absorption of bromelain on nasal administration at one-fourth oral dose. The enhanced cellular uptake and localization of bromelain in tissues of lung was observed significantly. Furthermore, faster onset and enhanced antihistaminic, bronchodilator and anti-asthmatic activity on bromelain's nasal administration signified faster absorption and higher in vivo stability of bromelain. Nasal administration significantly achieved decrease in level of oxidative and immunological markers along with restoration of antioxidant enzymes at considerably one-fourth dose administered orally. These findings distinctly manifested that nasal administration could be a substantial and effective route for bromelain delivery with enduring competency in asthma management.

MicroRNA expression profile of cholesterol metabolism analysis mediated by Thelenota ananas desulfated holothurin A saponin in RAW264.7 macrophage-foam cells.

Saponins from the sea cucumber, Thelenota ananas, have been reported to modulate cholesterol metabolism and may be useful in treating atherosclerosis and related diseases, but the mechanism remains unclear. This study is designed to investigate the effect of the saponin desulfated holothurin A (desHA), prepared from Thelenota ananas, on cholesterol and lipid metabolism in oxidized low-density lipoprotein (oxLDL)-induced RAW264.7 macrophage-foam cells. The detection and prediction of miRNAs were conducted by high-throughput sequencing and associated bioinformatics analysis. We found that desHA could play an essential role in the species and expression of miRNAs. There were more abundant miRNAs in the desHA-treated groups. miR-365-2-5p, -125b-1-3p, -744-5p, -330-3p, -125a-3p and -3057-5p were extremely suppressed by desHA under the oxLDL treatment, while miR-212-3p and miR-132-3p were significantly upregulated by desHA. The clusters of orthologous groups (COG) analysis indicated that the detected miRNAs participated in lipid transport and metabolism (I) and secondary metabolite biosynthesis, transport, and catabolism (Q), which were closely linked with cholesterol metabolism. The network of the nine miRNAs with higher degree of differential expression (miR-125a-3p, -23b-5p, -3057-5p, -330-3p, -365-2-5p and -744-5p, -345-5p -212-3p and -132-3p) and their target genes further showed relationships with inflammatory response, cell proliferation and cholesterol metabolism. Due to miR-125a-3p and miR-365-2-5p being involved in the regulation of more genes, which are mostly related to the functions involved in cholesterol metabolism, they may be potential targets for desHA to prevent or treat atherosclerosis.

Pineapple fruit improves vascular endothelial dysfunction, hepatic steatosis, and cholesterol metabolism in rats fed a high-cholesterol diet.

Hypercholesterolaemia is a significant risk factor for developing vascular disease and fatty liver. Pineapple (Ananas comosus), a tropical fruit widely cultivated in Asia, is reported to exhibit antioxidant and cholesterol-lowering activity; however, the potential hypolipidaemic mechanisms of pineapple fruit remain unknown. Therefore, we aimed to identify the anti-hypercholesterolaemic mechanism of pineapple fruit and to study the effect of pineapple fruit intake on hypercholesterolaemia-induced vascular dysfunction and liver steatosis in a high-cholesterol diet (HCD)-fed rats. Male Sprague Dawley rats were fed with standard diet or HCD, and the pineapple fruit was orally administered to HCD-fed rats for 8 weeks. At the end of treatment, vascular reactivity and morphology of aortas, as well as serum nitrate/nitrite (NOx), were determined. Liver tissues were also examined for histology, lipid content, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) activity, and protein expression of cholesterol metabolism-related enzymes. Results showed that pineapple fruit reduced the levels of hepatic cholesterol and triglycerides, and improved histological characteristics of a fatty liver in HCD-fed rats. Pineapple fruit also increased serum NOx, restored endothelium-dependent vasorelaxation, and reduced structural alterations in aortas of rats fed the HCD. In addition, a reduction of HMGCR activity and the downregulation of hepatic expression of HMGCR and sterol-regulatory element-binding protein 2 (SREBP2), as well as the upregulation of hepatic expression of cholesterol 7α-hydroxylase (CYP7A1) and LDL receptor (LDLR) were found in pineapple fruit-treated hypercholesterolaemic rats. These results indicate that pineapple fruit consumption can restore fatty liver and protect vascular endothelium in diet-induced hypercholesterolaemia through an improvement of hepatic cholesterol metabolism.

High-temperature ethanol fermentation from pineapple waste hydrolysate and gene expression analysis of thermotolerant yeast Saccharomyces cerevisiae.

High-temperature ethanol fermentation by thermotolerant yeast is considered a promising technology for ethanol production, especially in tropical and subtropical regions. In this study, optimization conditions for high-temperature ethanol fermentation of pineapple waste hydrolysate (PWH) using a newly isolated thermotolerant yeast, Saccharomyces cerevisiae HG1.1, and the expression of genes during ethanol fermentation at 40 °C were carried out. Three independent variables, including cell concentration, pH, and yeast extract, positively affected ethanol production from PWH at 40 °C. The optimum levels of these significant factors evaluated using response surface methodology (RSM) based on central composite design (CCD) were a cell concentration of 8.0 × 107 cells/mL, a pH of 5.5, and a yeast extract concentration of 4.95 g/L, yielding a maximum ethanol concentration of 36.85 g/L and productivity of 3.07 g/L. Gene expression analysis during high-temperature ethanol fermentation using RT-qPCR revealed that the acquisition of thermotolerance ability and ethanol fermentation efficiency of S. cerevisiae HG1.1 are associated with genes responsible for growth and ethanol stress, oxidative stress, acetic acid stress, DNA repair, the pyruvate-to-tricarboxylic acid (TCA) pathway, and the pyruvate-to-ethanol pathway.

AcCIPK5, a pineapple CBL-interacting protein kinase, confers salt, osmotic and cold stress tolerance in transgenic Arabidopsis.

Plant-specific calcineurin B-like proteins (CBLs) and their interacting kinases, CBL-interacting protein kinases (CIPKs) module, are essential for dealing with various biotic and abiotic stress. The kinases (CIPKs) of this module have been well studied in several plants; however, the information about pineapple CIPKs remains limited. To understand how CIPKs function against environmental cues in pineapple, the CIPK5 gene of pineapple was cloned and characterized. The phylogenetic analyses revealed that AcCIPK5 is homologous to the CIPK12 of Arabidopsis and other plant species. Quantitative real-time PCR (qRT-PCR) analysis revealed that AcCIPK5 responds to multiple stresses, including osmotic, salt stress, heat and cold. Under optimal conditions, AcCIPK5 gets localized to the cytoplasm and cell membrane. The ectopic expression of AcCIPK5 in Arabidopsis improved the germination under osmotic and salt stress. Furthermore, AcCIPK5 positively regulated osmotic, drought, salt and cold tolerance and negatively regulated heat and fungal stress in Arabidopsis. Besides, the expression of AcCIPK impacted ABA-related genes and ROS homeostasis. Overall, the present study demonstrates that AcCIPK5 contributes to multiple stress tolerance and has the potential to be utilized in the development of stress-tolerant crops.

Ectopic Overexpression of Pineapple Transcription Factor AcWRKY31 Reduces Drought and Salt Tolerance in Rice and Arabidopsis.

Pineapple (Ananas comosus (L.) Merr.) is an important tropical fruit with high economic value, and its growth and development are affected by the external environment. Drought and salt stresses are common adverse conditions that can affect crop quality and yield. WRKY transcription factors (TFs) have been demonstrated to play critical roles in plant stress response, but the function of pineapple WRKY TFs in drought and salt stress tolerance is largely unknown. In this study, a pineapple AcWRKY31 gene was cloned and characterized. AcWRKY31 is a nucleus-localized protein that has transcriptional activation activity. We observed that the panicle length and seed number of AcWRKY31 overexpression transgenic rice plants were significantly reduced compared with that in wild-type plant ZH11. RNA-seq technology was used to identify the differentially expressed genes (DEGs) between wild-type ZH11 and AcWRKY31 overexpression transgenic rice plants. In addition, ectopic overexpression of AcWRKY31 in rice and Arabidopsis resulted in plant oversensitivity to drought and salt stress. qRT-PCR analysis showed that the expression levels of abiotic stress-responsive genes were significantly decreased in the transgenic plants compared with those in the wild-type plants under drought and salt stress conditions. In summary, these results showed that ectopic overexpression of AcWRKY31 reduced drought and salt tolerance in rice and Arabidopsis and provided a candidate gene for crop variety improvement.

Genome-wide identification and characterization of the BBX gene family in pineapple reveals that candidate genes are involved in floral induction and flowering.

B-box zinc finger proteins contain one or two B-box domains, and sometimes, a CCT domain, which are involved in many biological processes, such as photomorphogenesis, flowering, anthocyanin synthesis and abiotic stress resistance. But the BBX gene family in pineapple has not been systematically studied. Nineteen BBX genes were detected in pineapple genome and divided into five groups according to phylogenetic analysis. The results of transcriptome analysis and RT-qPCR showed that most of AcBBX members were highly expressed during the flowering process, indicating that AcBBX gene may be involved in flower bud differentiation and morphogenesis. Transcriptional activation analysis showed that AcBBX6 and AcBBX18 had transcriptional activity and were located in the nucleus. Overexpression of AcBBX18 promoted flowering in Arabidopsis thaliana. These results provided a basis for further study functions and regulatory mechanism of BBX members in pineapple floral induction and flower development.